Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium

Import of DNA into mammalian nuclei is generally inefficient. Therefore, on e of the current challenges in human gene therapy is the development of eff icient DNA delivery systems. Here we tested whether bacterial proteins coul d be used to target DNA to mammalian cells. Agrobacterium tumefaciens, a pl ant pathogen, efficiently transfers DNA as a nucleoprotein complex to plant cells. Agrobacterium-mediated T-DNA transfer to plant cells is the only kn own example for interkingdom DNA transfer and is widely used for plant tran sformation, Agrobacterium virulence proteins VirD2 and VirE2 perform import ant functions in this process. We reconstituted complexes consisting of the bacterial virulence proteins VirD2, VirE2, and single-stranded DNA (ssDNA) in vitro. These complexes were tested for import into HeLa cell nuclei, Im port of ssDNA required both VirD2 and VirE2 proteins. A VirD2 mutant lackin g its C-terminal nuclear localization signal was deficient in import of the ssDNA-protein complexes into nuclei. Import of VirD2-ssDNA-VirE2 complexes was fast and efficient, and was shown to depended on importin alpha, Ran, and an energy source. We report here that the bacterium-derived and plant-a dapted protein-DNA complex, made in vitro, can be efficiently imported into mammalian nuclei following the classical importin-dependent nuclear import pathway. This demonstrates the potential of our approach to enhance gene t ransfer to animal cells.